Background/Objectives: Idiopathic pulmonary fibrosis (IPF) and other progressive fibrotic interstitial lung diseases (F-ILD) are characterised by progressive loss of lung function, worsening symptoms, and poor prognosis. Current antifibrotic therapies slow disease progression but do not arrest or reverse fibrosis and are frequently associated with adverse effects. Senicapoc, a selective KCa3.1 channel inhibitor, has shown antifibrotic effects in preclinical models, human lung myofibroblasts, and ex vivo human lung tissue. This study aims to determine whether senicapoc reduces the rate of decline in forced vital capacity (FVC) over 26 weeks, compared with placebo, in patients with IPF or other progressive F-ILD, while also characterising safety and tolerability. Methods: This is an investigator-initiated, prospective, randomised, double-blind, placebo-controlled, multicentre phase II trial. Adults with IPF or other F-ILD with documented progression despite optimised antifibrotic management according to standard care and individual tolerability will be randomised 1:1 to receive senicapoc 30 mg once daily or a matching placebo for 26 weeks in addition to standard care. The primary outcome is the rate of decline in FVC over 26 weeks. Secondary outcomes include changes in diffusion capacity, 6 min walk distance, dyspnoea, health-related quality of life, adverse events, and senicapoc plasma concentrations, with mortality and exacerbations assessed as exploratory outcomes. The planned sample size is 140 participants. The primary analysis will be conducted in the intention-to-treat population using a linear mixed-effects model for repeated measurements. Results: No results are available, as this article describes the study protocol. Conclusions: This study will provide proof of concept for the efficacy, safety, and tolerability of senicapoc in progressive fibrotic interstitial lung disease. If successful, it will support further clinical development of KCa3.1 inhibition as a novel antifibrotic strategy.

Severe asthma is a heterogeneous disease. The mechanisms driving airway pathology when type 2 (T2) cytokine activity is suppressed remain poorly understood. This study aimed to provide insight by identifying the airway molecular pathways of T2 biomarker-high and -low severe asthma. We analysed clinical and transcriptomic data from bronchial biopsies and brushes in the UK Refractory Asthma Stratification Programme multi-centre severe asthma cohort (18 corticosteroid-resistant T2 biomarker-high [T2-high], 23 T2 biomarker-intermediate [T2-intermediate], 11 T2 biomarker-low [T2-low]) plus 20 healthy controls pre- and post-treatment with high-dose inhaled corticosteroids (ICS). Many genes dysregulated in asthma vs. health were concordantly dysregulated in healthy subjects receiving ICS. Severe asthma as a whole, independent of confounding by ICS, was characterised by upregulation of mucins, CEACAM5, typical T2-genes (POSTN, CLCA1, CCL26), epithelial mast cell genes, and CPA4. T2-high severe asthma demonstrated upregulated T2-dependent genes, epithelial barrier and keratin genes, adaptive immune responses, and impaired ciliary function. T2-low asthma showed upregulated Th1- and IL-17-associated genes (IDO1, CXCL10, GBP1, LAG3), interferon-γ signalling, neuroimmune pathways, airway smooth muscle-related genes, and neutrophil enrichment. T2-intermediate asthma exhibited a mixed molecular profile sharing features of T2-high and T2-low endotypes, with selective expression of the pathogen defence and antiviral response genes. The results were validated using bronchoscopy data from the U-BIOPRED Consortium. This study defines airway molecular endotypes of severe asthma associated with T2 biomarker high and low phenotypes, independent of corticosteroid effects. These findings offer insights for severe asthma management and the development of targeted biologic therapies.

Abstract Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease characterised by aberrant fibroblast function, extracellular matrix (ECM) remodelling and defective tissue repair. Protein kinase N2 (PKN2) is associated with accelerated forced vital capacity decline in IPF, but its functional role in pulmonary fibrosis remains unknown. We hypothesised that PKN2 regulates fibroblast phenotype and tissue repair. Methods PKN2 expression was assessed in human lung tissue, induced sputum and primary airway and parenchymal fibroblasts from non-fibrotic controls and patients with interstitial lung disease (ILD). DNA methylation was profiled using the Illumina HumanMethylationEPIC array. PKN2 function was investigated by siRNA-mediated depletion in primary human lung fibroblasts using transcriptomic, proteomic and functional analyses. Tissue repair was assessed following pharmacological PKN inhibition in zebrafish. Results PKN2 expression was reduced in ILD lung tissue and primary airway and parenchymal fibroblasts and further suppressed by TGF-β1. Differential methylation was identified across the PKN2 locus in both fibroblast populations. Integrated transcriptomic and proteomic profiling following PKN2 depletion revealed coordinated remodelling of ECM, cell adhesion, non-canonical WNT and VEGF pathways, including dysregulation of COL1A1, WNT, VEGF and MMP1. PKN2 loss increased VEGF and MMP-1 secretion and accelerated fibroblast wound closure. PKN inhibition altered epithelial organisation and collagen fibre alignment during zebrafish wound repair. Conclusion PKN2 loss drives fibroblast reprogramming and aberrant ECM remodelling, establishing PKN2 as an important regulator of pulmonary fibroblast homeostasis and tissue repair.

KCa3.1 encodes the intermediate-conductance calcium-activated potassium channel KCa3.1, a regulator of membrane potential and calcium-dependent signalling in cardiovascular and immune cells. Increasing evidence indicates that KCa3.1 is a shared driver of vascular remodelling, inflammation, fibrosis, and electrical instability across multiple cardiovascular diseases. In ischaemic heart disease (IHD), KCa3.1 is upregulated in endothelial cells, vascular smooth muscle cells, macrophages, and T lymphocytes, where it promotes smooth muscle proliferation, neointimal formation, and chronic vascular inflammation. Genetic deletion or pharmacological blockade of KCa3.1 reduces atherosclerotic plaque burden and restenosis in animal models. In atrial fibrillation (AF), KCa3.1 contributes to electrical remodelling by shortening atrial action potential duration and to structural remodelling by driving fibroblast activation and collagen deposition. KCa3.1 also regulates macrophage polarisation and pro-inflammatory cytokine release in atrial tissue, linking immune activation to arrhythmogenic substrate formation. Inhibition of KCa3.1 prolongs atrial refractoriness, attenuates atrial fibrosis, and reduces AF inducibility in multiple preclinical models. Emerging data in valvular heart disease suggest that KCa3.1 is upregulated in valvular interstitial cells and regions of active calcification, where it supports myofibroblast differentiation, osteogenic signalling, and inflammatory crosstalk, implicating the channel in fibrocalcific valve degeneration. Collectively, these findings position KCa3.1 as a central molecular integrator of electrical, fibrotic, and inflammatory pathways in cardiovascular disease. The availability of selective KCa3.1 inhibitors with established human safety profiles supports the feasibility of therapeutic translation. Targeting KCa3.1 may enable disease-modifying strategies that extend beyond symptom control to suppress maladaptive cardiovascular remodelling.

Abstract Introduction Aortic stenosis (AS) is characterised by progressive aortic valve (AV) leaflet fibrosis and calcification, yet no medical therapies exist to slow disease progression. AV interstitial cells (VICs) that differentiate into myofibroblasts are central drivers of fibrosis. The Ca 2+ -activated K + channel K Ca 3.1 promotes pro-fibrotic signalling in several fibrotic diseases, however its role in AS remains unknown. Methods K Ca 3.1 protein expression was examined in paraffin embedded tissue by Immunohistochemistry from control and AS valve tissue. VICs were isolated, cultured and phenotypically characterised as myofibroblasts from AV tissue obtained from patients with severe tricuspid AS undergoing surgical AV replacement (n=19). K Ca 3.1 mRNA and protein expression were assessed by qRT-PCR and immunohistochemistry, and functional channel activity confirmed using patch-clamp electrophysiology. The effects of transforming growth factor-β1 (TGFβ1) stimulation and pharmacological inhibition with the selective K Ca 3.1 blocker senicapoc were examined. Results Immunoreactive K Ca 3.1 channels and smooth muscle actin were detected in both control and AS aortic valve tissue, localised to elongated, nucleated interstitial cells, with significantly higher expression observed in AS tissue compared to control. Isolated VICs exhibited an activated myofibroblast phenotype, expressing THY-1, vimentin, collagen and α-smooth muscle actin (αSMA) (n=9). Myofibroblasts expressed K Ca 3.1 mRNA and protein and demonstrated functional plasma membrane channels. TGFβ1 stimulation increased K Ca 3.1, αSMA and collagen type I mRNA expression, while K Ca 3.1 blockade with senicapoc (100 nM) significantly attenuated TGFβ1-induced αSMA expression, stress fibre formation and collagen gel contraction. Senicapoc had no effect on myofibroblast proliferation or migration. Conclusions We show for the first time that functional K Ca 3.1 channels are expressed in human AS tissue and AV myofibroblasts, where they regulate myofibroblast contraction, α-SMA expression, and differentiation, promoting pro-fibrotic activity. These responses are attenuated by the selective K Ca 3.1 inhibitor senicapoc. Given its established safety in phase 3 clinical trials, K Ca 3.1 inhibition represents a promising and readily translatable anti-fibrotic therapeutic strategy for AS.

BACKGROUND:Asthma symptoms often guide disease assessment and management, but their prognostic and predictive value is unclear. We evaluated the extent to which symptom burden measured by the 5-item Asthma Control Questionnaire (ACQ-5) predicts future severe asthma attacks and response to anti-inflammatory therapy. METHODS:We conducted an individual participant data meta-analysis of selected randomised controlled trials and translational observational cohort studies of asthma of varying severity. Primary analyses used the ORACLE2 patient-level meta-analysis (n=6513) of control-group participants from 22 randomised controlled trials. Additional datasets from studies assessing type 2 targeting anti-inflammatory therapies were included on the basis of availability of data, to provide insight into the association between ACQ-5 and sputum cell counts or mediator data. Additional datasets were the DREAM intravenous mepolizumab group (n=461); a cross-sectional severe asthma cohort (SA-OT, n=74); and two acute asthma cohorts (PRISMA, biologic-naive, n=53; BOOST, anti-interleukin-5-treated, n=60). Associations between baseline ACQ-5 scores and clinical, physiological, and inflammatory profiles, asthma attack risk, and anti-inflammatory treatment responses were examined. FINDINGS:Across five datasets encompassing 7161 distinct participants, asthma severity, lung function, inflammatory profiles, comorbidities, and ACQ-5 results varied widely. The proportion of patients with high symptom burden (ACQ-5 score >1·5) ranged from 39% to 100%. Baseline ACQ-5 showed no consistent cross-sectional association with other clinical, physiological, or inflammatory asthma features. Each 0·5-point increase in baseline ACQ-5 score was associated with a modest increase in future asthma attack risk (adjusted rate ratio [aRR] 1·09 [95% CI 1·06-1·12]; Δ R2=0·02 vs multivariable prediction model without ACQ-5). Baseline ACQ-5 score did not alter relative and absolute attack risk reduction from intravenous mepolizumab in DREAM. By contrast, patients with high blood eosinophil counts and high fractional exhaled nitric oxide (FeNO) had the highest relative and absolute risk reduction (2·81 vs 1·17 attacks; aRR 0·38 [95% CI 0·25-0·57]). In the PRISMA and BOOST datasets, ACQ-5 was not associated with post-corticosteroid lung function change. Across studies, relative and absolute treatment effects showed consistent associations with blood eosinophil counts and FeNO. INTERPRETATION:In a large, individual patient-level meta-analysis of randomised controlled trials and translational prospective observational cohort studies, we observed little alignment of symptom burden with other clinical, physiological, or biological features of asthma and modest prognostic value for severe attacks. Conversely, type 2 biomarkers more reliably identified patients at high risk and those likely to respond to treatment. Symptoms might require contextual interpretation to guide anti-inflammatory escalation in asthma. FUNDING:National Institute for Health and Care Research, Association Pulmonaire du Québec, Fonds de Recherche du Québec-Santé, and The Academy of Medical Sciences.

ABSTRACTBackgroundAirway remodelling is a feature of severe asthma with airway epithelial damage observed frequently. We evaluated the role of WNT5a and TGF‐β1 in asthmatic airway biopsies and in sputum and bronchial brushings assessed their role in remodelling.MethodsWNT5a and TGF‐β1 protein expression were assessed in the lamina propria epithelium of people with asthma (GINA 1–3, n‐8 and GINA 4–5, n‐14) and healthy subjects (n‐9), alongside relevant remodelling markers. The effects of WNT5a and TGF‐β1 on BEAS‐2B epithelial cell wound healing and differentiation were assessed in vitro. Replication was performed in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U‐BIOPRED) study in sputum (n = 120) and bronchial brushes (n = 147).ResultsWNT5a and TGF‐β1 protein expression were significantly increased in the airway epithelium and lamina propria in asthma patients with concurrent airflow limitation or severe disease. Furthermore, WNT5a protein expression in the lamina propria correlated with tissue eosinophils and vascular remodelling. Airway epithelial WNT5a was co‐localised predominantly to airway basal cells and correlated with Th17 gene expression (r = 0.40, p = 0.025) and both the % intact (rs = 0.54, p = 0.001) and % denuded epithelium (rs = −0.39, p = 0.003). Experiments in BEAS‐2B cells confirmed that WNT5a at maximal physiological concentrations (1 μg/mL), promoted epithelial wound healing, independently of TGF‐β1, as well as induction of EMT‐like morphology. WNT5a mRNA was associated with severe asthma, airflow limitation, sputum eosinophilia and Th2, and Th17 and neutrophil activation transcriptomes in sputum in U‐BIOPRED.ConclusionWNT5a is associated with both airway remodelling and severe asthma.Trial RegistrationClinicalTrials.gov identifier: NCT01982162

BACKGROUND:The optimal approach to weaning maintenance oral corticosteroids (mOCS) in patients with severe asthma receiving biologics remains unclear. Previous studies assessed hypothalamic-pituitary-adrenal function at 5 mg daily prednisolone, a supraphysiologic dose for many, necessitating further mOCS reduction for adrenal recovery. OBJECTIVE:We evaluated a protocol-driven, nurse-led mOCS withdrawal pathway with clinical oversight for patients with severe asthma receiving biologics. METHODS:Patients with severe asthma receiving biologics, who had reduced mOCS to 5 mg prednisolone daily and maintained good asthma control, entered the withdrawal pathway. Prednisolone was decreased to 4 mg daily for 6 weeks and then 3 mg daily for 6 weeks, followed by 09.00 serum cortisol measurement. Patients with cortisol greater than 25 nmol/L followed a 20-week weaning protocol. Serum cortisol was rechecked 12 weeks after stopping mOCS. RESULTS:Of 102 patients, 92 had cortisol greater than 25 nmol/L with 3 mg prednisolone and continued weaning. A total of 73 (72%) successfully discontinued mOCS with median (interquartile range) cortisol increasing from 192 (88-299) nmol/L with 3 mg prednisolone to 314 (248-437) nmol/L at 12 weeks after discontinuation (P < .0001). Twenty-nine patients (28%) paused weaning owing to adrenal insufficiency symptoms (n = 22), worse asthma control (n = 1), anxiety (n = 2), and other reasons (n = 4). The baseline cortisol in this group was 53 (25-166) nmol/L, and patients are currently well and receiving a median of 3.0 (3.0-3.9) mg prednisolone. Duration of prior oral corticosteroid use was significantly shorter in the group that was successfully weaned compared with those who failed weaning (P = 0.003). No serious adverse events occurred. CONCLUSION:Most clinically stable patients with asthma receiving biologics successfully withdrew mOCS without requiring dynamic adrenal function testing.

INTRODUCTION:Novel treatments for idiopathic pulmonary fibrosis (IPF) are needed urgently. A better understanding of the molecular pathways activated by TGFβ1 in human lung tissue may facilitate the development of more effective anti-fibrotic medications. This study utilized proteomic analysis to test the hypothesis that TGFβ1 induces pro-fibrotic effects on human lung parenchyma proteome, and to evaluate the viability of this model for testing novel therapeutic targets. METHODS:Non-fibrotic human lung parenchymal tissue from 11 patients was cultured for 7 days in serum-free (SF) media supplemented with TGFβ1 (10 ng/mL) or vehicle control, and the putative antifibrotic KCa3.1 ion channel blocker senicapoc or vehicle control. The tissue was homogenised, digested for bottom-up proteomics, and analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Principal component analysis, differential expression analysis, pathway analysis, and drug repurposing analysis were performed. RESULTS:TGFβ1 stimulation for 7 days induced a strong fibrotic protein response relevant to IPF pathology. A total of 2391 proteins were quantified, 306 upregulated and 285 downregulated (FDR-adjusted p-value<0.05). Of these, 118 were upregulated and 28 downregulated at log2(FC) > 0.58. These changes were attenuated by senicapoc (100 nM). Drug repurposing analysis identified 265 drugs predicted to inhibit the effects of TGFβ1 in this model. These included clotrimazole, a KCa3.1 blocker, and nintedanib, a drug licenced for the treatment of IPF, providing validation of this approach. CONCLUSION:A pro-fibrotic proteome is induced in human lung parenchyma exposed to TGFβ1, sensitive to pharmacological intervention. This approach has the potential to enhance therapeutic drug screening for IPF treatment.

Background:∼5-10% of patients with asthma have severe disease. A proportion remain symptomatic despite suppression of T2-related inflammation but what drives persistent symptoms remains unclear. Eicosanoids exert a functional role in pulmonary inflammation. We explored the relationship between urinary eicosanoids, asthma symptoms, obesity and T2-biomarker status. Methods:Urine was sampled during a randomised controlled trial assessing corticosteroid optimisation using T2-biomarker directed care at scheduled study visits (n=728) and at exacerbation (n=103). Urine eicosanoid concentrations were measured by mass spectrometry, then log2-transformed, z-scored and concatenated by biosynthetic pathway generating six pathway scores. Results were stratified by T2 status (T2-low: exhaled nitric oxide fraction (F ENO) <20 ppb and blood eosinophil count (BEC) <0.15×109 cells·L-1; versus T2-high: F ENO ≥20 ppb and BEC ≥0.15×109 cells·L-1), symptoms (symptom-low: Asthma Control Questionnaire-7 (ACQ-7) <1.5; versus symptom-high: ACQ-7 ≥1.5) and obesity. Results:Isoprostane (pathway score p=0.02) and thromboxane (pathway score p=0.04) levels were higher in symptom-high versus symptom-low, T2-low participants. Isoprostane levels were greater in symptom-high versus symptom-low participants, irrespective of T2 status (pathway score p=0.01). Cysteinyl-leukotriene E4 levels (LTE4) were elevated in T2-high versus T2-low participants (pathway score p=0.0007), irrespective of symptoms. Corticosteroid exposure, obesity and exacerbations were not associated with increased eicosanoid levels (p≥0.05). Conclusion:Raised urinary eicosanoid levels of isoprostanes and thromboxanes were associated with increased symptoms in T2-low severe asthma. Elevated excretion of these metabolites in T2-low participants could reflect increased thromboxane-receptor (TP) activation, which may be promoting increased asthma severity and bronchoconstriction. Further research and interventions are needed to explore the role of TP modulation in T2-low severe asthma.

Introduction: Patients with type 2 (T2) low airway disease such as asthma poorly respond to current inhaled corticosteroid therapy and biologics targeting T2 inflammation. In addition, T2 low airways are prone to be colonized by bacterial pathogens. Fractional exhaled nitric oxide (FeNO) is an established biomarker for T2 inflammation based on IL-4Rα-mediated upregulation of nitric oxide synthase (NOS2) and subsequent NO release by epithelial cells. Conversely, pharmacologic inhibition of IL-4Rɑ signaling in asthma reduces FeNO. We have identified a mutually exclusive relationship between elevated FeNO levels and high airway load of Haemophilus influenzae in asthmatic patients with frequent exacerbations. Given that nasal epithelial NO production mediates bacterial killing in co-culture experiments in vitro we hypothesized that bronchial epithelial NO has a direct antimicrobial function and underlies the microbial and FeNO relationship. We examined whether bacterial killing is enhanced by acute NOS2 induction mediated by IL-13 treatment of normal human bronchial epithelial cells (NHBECs). Methods: Primary NHBECs differentiated at air-liquid interface (ALI) culture were stimulated with IL-13 (20 ng/mL) for 24 h. A non-typeable strain of H. influenzae (NTHi) was co-cultured on the apical surface of terminally differentiated NHBECs for 4 h with or without an iNOS pharmacological inhibitor, L-NAME. Bactericidal activity of NHBECs was assessed by flow cytometry-based BacLight assay and was further validated by counting colony forming units (CFU) plated after co-culture. Expression of T2 signature genes in NHBECs was studied using qPCR. Results: Acute treatment of fully differentiated NHBECs with IL-13 induced a significant upregulation of NOS2, along with T2 gene signature genes, POSTN, SERPINB2, and CLCA1. This condition was associated with increased extracellular antibacterial potential of NHBECs as evident by 8-fold reduction in CFU count of NTHi and elevation of dead bacterial population in flow cytometry analysis. Mechanistically, IL-13-induced antibacterial activity of NHBECs was suppressed upon iNOS blockade, by L-NAME, suggestive of an NO-dependent antibacterial mechanism of action. IL-13 and L-NAME did not directly affect planktonic growth of NTHi in the absence of NHBECs suggestive of no direct bactericidal activity of IL-13 on this pathogen. Conclusions: Bronchial epithelial release of extracellular NO mediated by IL-4Rɑ signaling has antimicrobial function in vitro and may mechanistically underlie NTHi colonization in T2/FeNO low airway disease contexts. These data provide a potential mechanistic basis for bacterial colonization for intrinsic and pharmacologically induced T2/FeNO low contexts and support that the assessment of specific bacteria-host interactions could influence clinical decision-making in asthma.

Idiopathic pulmonary fibrosis (IPF) is a severe lung disease affecting around 5 million people globally, with a median survival of 3-4 years. Characterized by excessive scarring of lung tissue, IPF results from the accumulation of myofibroblasts that deposit extracellular matrix (ECM), causing fibrosis. Current treatments, pirfenidone and nintedanib, slow the disease but do not stop its progression. IPF pathogenesis involves repeated alveolar injury, leading to pro-fibrotic mediators like TGFβ1, which trigger fibroblast-to-myofibroblast transitions and ECM deposition.Recent research suggests that transient receptor potential (TRP) channels, such as TRPV4, TRPC6, and TRPA1, play a key role in regulating calcium signalling and mechanical stress, crucial in myofibroblast activation.Targeting TRP channels may disrupt fibrosis and offer new therapeutic strategies. Preclinical studies indicate that inhibiting TRP channels could reduce fibrosis, warranting further trials to explore their efficacy and safety in treating IPF and related fibrotic conditions

Airway hyperresponsiveness (AHR) is a key clinical feature of asthma. The presence of AHR in people with asthma provides the substrate for bronchoconstriction in response to numerous diverse stimuli, contributing to airflow limitation and symptoms including breathlessness, wheeze and chest tightness. Dysfunctional airway smooth muscle (ASM) significantly contributes to AHR and is displayed as increased sensitivity to direct pharmacological bronchoconstrictor stimuli, such as inhaled histamine and methacholine (direct AHR), or to endogenous mediators released by activated airway cells such as mast cells (indirect AHR). Research in in vivo human models has shown that the disrupted airway epithelium plays an important role in driving inflammation that mediates indirect AHR in asthma, through the release of cytokines such as TSLP and IL-33. These cytokines upregulate type 2 cytokines promoting airway eosinophilia and induce the release of bronchoconstrictor mediators from mast cells such as histamine, prostaglandin D2 and cysteine leukotrienes. While bronchoconstriction is largely due to ASM constriction, airway structural changes termed ‘remodelling’, likely mediated in part by epithelial-derived mediators, also lead to airflow obstruction and may enhance AHR. In this review, we outline the current knowledge of the role of the airway epithelium in AHR in asthma and its implications on the wider disease. Increased understanding of airway epithelial biology may contribute to better treatment options, particularly in precision medicine.